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In Vitro–Derived Gametes from Stem Cells
Franklin D. West, PhD1 Reza Shirazi, PhD2 Pezhman Mardanpour, PhD4 Servet Ozcan, PhD5,6
Gokcen Dinc, PhD6,7 Dewey H. Hodges, PhD4 Hamid Reza Soleimanpour-Lichaei, PhD3
Karim Nayernia, PhD6,8
Georgia
2 Department of Anatomical Sciences, School of Medicine, Qazvin
University of Medical Sciences, Qazvin, Iran
3 National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
4 Georgia Institute of Technology, Guggenheim School of Aerospace
Engineering, Atlanta, Georgia
5 Department of Biology, Faculty of Science
6 Genome and Stem Cell Research and Application Centre
7 Department of Microbiology, Faculty of Medicine, Erciyes University,
Kayseri, Turkey
8 GENEOCELL, Advanced Molecular and Cellular Technologies,
Montreal, Canada
Address for correspondence and reprint requests Karim Nayernia,
PhD, GENEOCELL, Advanced Molecular and Cellular Technologies,
Montreal, Canada (e-mail: [email protected]).
Semin Reprod Med 2013;31:33–38
Abstract
Keywords
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infertility
stem cells
germ cells
IVD gametes
Sperm and eggs are essential cells for reproduction and fertility in mammals. Lack of
sperm production is one of the leading causes of infertility, a major and growing
problem in the developed world affecting 13 to 18% of reproductive-age couples. The
birth of the first test tube baby by in vitro fertilization marked an advance in infertility
treatment. Later on, several important new techniques called assisted reproductive
technologies were developed to help couples who experience infertility. One limiting
factor is the requirement of reproductive cells (gametes) for use in in vitro fertilization.
For azoospermic men lacking sperm cells, producing gametes in vitro could be a new
window to overcome infertility. In the past few years, several reports have been
published on generating germ cells from stem cells, one of the epitomes of which
was the report on functional in vitro–derived (IVD) germ cells. These mature haploid
sperm cells from mouse embryonic stem cells were capable of egg fertilization and
producing live offspring. In tandem with previous advancements in germ cell research,
development of new technologies based on IVD gametes will change the future of
infertility and provide a new basis for the establishment of novel therapeutic approaches
to cure more complicated conditions of infertility. In addition, IVD gametogenesis
provides an accessible system for studying the specification and differentiation of sperm
cells and related processes such as meiosis, morphogenesis, and motility.
Infertility is a major and growing problem in the developed
world affecting 13 to 18% of reproductive-age couples.1
A series of conditions may lead to infertility including decreased sperm counts, increasing age of first-time parents,
increase in the incidence of polycystic ovary syndrome, and
the rising incidence of sexually transmitted diseases.2 The
Issue Theme Stem Cells Helping
Reproductive Medicine; Guest Editor,
Carlos Simón, MD, PhD
birth of the first test tube baby by in vitro fertilization in 1978
marked an advance in infertility treatment. Later on, several
important new techniques, collectively called human assisted
reproductive technologies (ART), were developed to help
couples who experience infertility.3 However, some patients
devoid of germ cells such as azoospermia patients cannot
Copyright © 2013 by Thieme Medical
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Tel: +1(212) 584-4662.
DOI http://dx.doi.org/
10.1055/s-0032-1331795.
ISSN 1526-8004.
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1 University of Georgia, Regenerative Bioscience Center, Athens,
In Vitro–Derived Gametes from Stem Cells
West et al.
benefit from ART treatments. Several approaches have been
developed to obtain an alternative source of normally developed spermatozoa and oocytes including in vitro–derived
(IVD) gametes for infertile couples. Recently two main techniques for producing gametes in vitro, somatic cell haploidization (nuclear transfer, cloning) and germ cell
differentiation from embryonic stem cells (ESCs), were successfully developed in mammals, opening a potential new
window for the treatment of human sterility.3 During haploidization, somatic cells (2N) go through induced meiosis,
and the diploid chromosomes are reduced to a haploid (1N)
state to form gametes.4 Haploidization can be achieved by
introducing a diploid somatic cell into cytoplasm that is
preprogrammed to undergo meiosis. These haploid (1N) cells
can then be potentially utilized as an infertility treatment.3,4
There is an increasing interest by scientists and clinicians
in applying stem cell technologies in the reproductive field, in
particular generating gametes in vitro.5–30 Much of the
interest has centered on the creation of functional gametes
from stem cells to study the complicated processes that occur
during germ cell development and applying this technology
in clinical treatment. New research offers promising developments in establishing novel stem cell–based approaches for
the generation of germ cells in various stages of differentiation from mice and human stem cells, from both embryonic
and nonembryonic cells.
It has been shown that ESCs are able to differentiate
spontaneously and rapidly into germ cells of various stages
following induction utilizing a myriad of culture systems. It
appears that IVD germ cell development occurs in a similar
manner as their in vivo derived counterparts.5–8 More surprisingly, scientists could report live offspring following the
injection of IVD sperm-like cells into oocytes.8 This demonstrated the possibility of deriving functional and mature
gametes from ESCs. The exact mechanism by which gamete-like cells are generated during stem cell culture is still
unclear.
There is some evidence that germ cells can be obtained
from adult stem cells residing in bone marrow, peripheral
blood cells, and fetal skin cells This could indicate that adult
tissue-derived stem cells may provide a new source for in
vitro produced germ cells. However, derivation of fully differentiated gametes from non-ESCs has not yet been reported. In addition, recent studies have shown that stem
cells in bone marrow and peripheral blood may serve as a
reservoir for new gametes in mouse ovaries and that reprogramming may not be required.9,10 Having a pool of stem cells
capable of giving rise to germ cells in organs like bone marrow
or peripheral blood may one day make it possible to delay the
age of normal menopause and preserve fertility in female
cancer patients where chemotherapy often leads to gamete
damage.10
In this review after a short introduction on germ cell
biology and development, we discuss in vitro derivation of
germ cells in various stages, from primordial germ cells to
fully developed gametes, from embryonic and nonembryonic
stem cells, and the differences in generating male and female
gametes.
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In Vitro–Derived Germ Cells from ESCs
Hübner et al published the first report of IVD germ cells in
2003.5 They demonstrated that mouse ESCs (mESCs), both
male and female, in culture can differentiate into female germ
cells precursors that can begin meiosis and then form folliclelike structures. In vitro oocyte-like cell derivation without
specific changes to culturing condition suggested spontaneous differentiation of germ cells from ESCs. In this study,
mESCs were transfected with a vector harboring a modified
version of oct4 promoter to drive a germ cell–specific expression of green fluorescent protein (GFP).5
Shortly after the first in vitro demonstration of ESC
differentiated into oocytes, the first attempt to produce
male germ cells was performed by Toyooka et al.6 mESCs
were aggregated into EBs, which are three-dimensional
structures that induce differentiation and mimic the early
embryo environment producing cell types of the three germ
layers (ectoderm, endoderm, and mesoderm). They also
obtained mouse vasa homolog (Mvh), a robust germ cell–
specific marker, positive cells from mESCs through embryoid
body (EB) formation. Using a bone morphogenetic protein
(BMP) 4 producing M15 cell line as feeder cells resulted in
enhanced germ cell formation within EBs. To track and isolate
germ cells in culture, the Mvh gene was tagged with GFP. GFPpositive cells were sorted by fluorescent-activated cell sorting
(FACS) and co-cultured with gonadal cells, followed by transplantation beneath the testis capsule of male mice to examine
further in vivo development of newly formed germ cells.
Implanted cells showed engraftment into the lumens of
seminiferous tubule-like structures distinct from those of
the host testis. Some of these cells gave rise to morphologically normal spermatozoa. However, functional data concerning the fertilization capacity of IVD male germ cells upon
injection into donor oocytes was still needed to determine the
full potential of these cells.6
Geijsen et al also developed a system capable of inducing
spontaneous differentiation of primordial germ cells (PGCs)
from mouse ESCs through EB formation.16 Adding retinoic
acid (RA), a growth factor produced in the testis that promotes PGC proliferation, enhanced mESCs differentiation into
SSEA-1þ cells, which were then magnetic-activated cell
sorted based on the SSEA-1 markers. The authors showed
that IVD germ cells displayed erasure of the methylation
pattern in some imprinted loci, a hallmark of normal germ
cell development.6 These cells were further differentiated
into postmeiotic germ cells and formed haploid cells. Intracytoplasmic injection of sorted cells into oocytes led to the
biological activation of eggs and the formation of blastocysts
in 20% of the cases of fertilized oocytes. It was not tested if the
embryos were capable of developing normally with uterine
transfer.16
Taking a step forward, Clark et al showed for the first time
that human ESCs (hESCs) were capable of differentiating into
early germ cells.7 These investigators utilized EB differentiation of female and male hESCs to produce mixed-cell populations spontaneously. A subset of these differentiated cells
expressed markers belonging to different stages of germline
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cells such as Stella, DAZL, and c-Kit. Furthermore, the expression levels of the primordial germ cells marker mouse vasa
homolog (Mvh) and meiotic marker SYCP3 were increased in
EBs. Collectively, the results of this study showed hESCs of
both sexes spontaneously form putative germ cells and
undergo advanced stages of germ cell development entering
into meiosis. Interestingly, it was shown that germline-associated genes such as DAZL were expressed in undifferentiated
hESCs. This suggested that a subpopulation of hESCs may not
be truly undifferentiated and may contain some cells committed to the germ cell lineage.7
Efforts to derive female germ cells continued with a study
by Lacham-Kaplan et al in 2006 in which ESC-derived EBs
were cultured in conditioned medium obtained from testicular cell cultures of newborn male mice.11 This unique
culturing system led to the generation of oocyte-like cells
enclosed within follicular-like structures with one or two
layers of flattened cells that expressed oocyte-specific
markers such as ZP3. Further characterization of these cells
is required to determine the detailed morphological structure
of the zona pellucida. It is also necessary to determine the
specific stage of derived germ cells and further examine their
functionality.11
Novak et al explored the ability of male mESCs to differentiate into germ cells.12 They report the successful derivation
of follicle-like structures similar to those seen by Hübner
et al.5,12 Although the expression of meiotic proteins SYCP3,
SYCP1, SYCP2, and Rec8 were reported, surprisingly, formed
germ cells were not able to continue through meiosis. These
IVD cells showed no signs of synapsis, recombination, or
appropriate segregation of chromosomes, which are all key
elements of early meiosis.12
Qing et al explored a novel two-step approach to derive
female germ cells from mESCs.13 First they obtained PGCs
through EB formation followed by a second step where EBs
were co-cultured with a fetal ovarian granulose layer. Derivation of PGCs was detected by analysis of PGC-specific
markers. An increase in gene expression of the meiotic
marker SYCP3 and oocyte-specific markers such as GDF9
were reported in differentiated cells. The GDF9-positive cells
resembled an immature oocyte without zona pellucida
formation.13
Chen et al also reported the development of a ovarian
follicle-like structure from hESCs through EB formation.14
Detection of c-KIT and low amounts of Mvh was observed.
However, it seems that three-dimensional EB formation is not
essential for oocyte-like formation.14
Kerkis et al reported the derivation of both male and
female germ cells from mESCs through EB formation with
the addition of RA.15 Depending on the length of culture time,
they showed that male germ cells appeared in the medium.
Then the production of sperm-like cells could induce female
germ cell generation. Interestingly, IVD sperm could penetrate and fertilize an IVD oocyte, leading to structures resembling blastocysts.15
Another study using mESCs transfected with a reporter
gene (GFP) associated with GDF9 gene promoter, a female
germ cell marker, through EB and monolayer differentiation
West et al.
was performed by Salvador et al.16 Cells rapidly became GFPþ
and displayed an oocyte phenotype.
The first report of functional sperm derived in vitro was
shown by Nayernia et al using a two-step sorting method
after monolayer differentiation of mESCs.8 They reported the
production of live offspring from IVD sperm. PGCs were
obtained from mESCs with a Stra8-driven GFP reporter after
germ cell induction with RA. These cells were then isolated by
FACS. Sorted cells were then grown in RA-supplemented
medium and transfected with a second reporter, Prm1DsRed. Using a two fluorescent reporter system enabled
them to track the progression of mESC-derived germ cells
through the spermatogenic process and isolate cells with a
sperm-like morphology. The injection of sorted sperm-like
cells resulted in blastocysts and pregnancies after transferring into uterus. Most of the fertilized eggs appeared to die,
yet a few offspring were born. However, due to developmental complications presumably because of the mESC to IVD
germ cell differentiation process, mouse pups died prematurely. This result suggests that epigenetic reprogramming of
the derived sperm-like cells may not have been complete.
Generation of Germ Cells from Non-ESCs
Several reports have demonstrated that adult stem cells are
able to differentiate into early germ cells. In mouse, an
elevated expression of Mvh has been observed in bone
marrow–derived mesenchymal stem cells treated with
BMP4.25 However, differentiation of bone marrow cells to
germ cells is restricted to early stages of germ cell development with additional factors likely being required to obtain
more mature cells.26 Similar germ cell differentiation potential has been shown in human bone marrow–derived mesenchymal stem cells.27 In addition to germ cells, Lue et al
observed differentiation of bone marrow–derived mesenchymal stem cells to testicular somatic cell lineages,28 which
raises novel possibilities for the treatment of male infertility
in human by stem cell–based therapeutic approaches.27,29
Generation of germ cells and gametes from embryonic stem
cells is summarized in ►Fig. 1.
Outlook
Improvement of IVD Germ Cell Technology
One of the major concerns in the clinical application of IVD
germ cells is the potential of those cells to produce healthy
progeny. Mice produced by sperm cells derived from ESCs
showed health problems due to inappropriate reestablishment of paternal imprinting genes during gametogenesis and
embryogenesis.8 However, this problem potentially has been
overcome with a recent study using multipotent adult germ
cells (maGSCs) derived from testis to produce IVD sperm
cells.30 The differences in the epigenetic state of gametes
derived from ESCs and maGSCs may be due to the starting
populations, with ESCs carrying somatic imprinting versus
the germ cell imprinting of maGSCs. In the normal physiologic
state of an early mouse embryo, PGCs are formed in response
to BMP signaling and activation of the germ cell gene
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In Vitro–Derived Gametes from Stem Cells
West et al.
Figure 1 Schematic representation of in vitro–derived (IVD) (A) female and (B) male germ cells and gametes from embryonic stem cells. EB,
embryoid body; FACS, fluorescent-activated cell sorting; ICSI, intracytoplasmic sperm injection; PGC, primordial germ cell; RA, retinoic acid.
expression.31 These cells, still having somatic imprinting,
migrate in the embryo through the dorsal mesentery and
hindgut where they undergo imprinting erasure shortly
before arriving at the genital ridge. This erasure of somatictype imprinting is important in PGCs because these cells
require germ cell specific imprinting to pass on to the next
developing embryo. In male mice, reinstatement of imprinting starts in pro-spermatogonia at embryonic day 13.5
(E13.5). In addition, these cells undergo retrotransposon
repression to safeguarding genome integrity by virtue of a
subset of DNA- and histone-modifying enzymes and piwi
family proteins. In marked contrast with male germ cells
undergoing genome imprinting well before meiotic activity,
the female germ cells start this epigenetic process during
meiosis around prophase I.31 In contrast with their male
counterparts, female germ cells do not require the activity of
the genome-safeguarding proteins because their mitotic
proliferation is very limited relative to the massive proliferation activity of spermatogonial germ cells after puberty.31
Therefore the normal molecular events of imprinting need to
be accurately recapitulated in any in vitro technique aiming at
generating IVD gametes to culminate in the birth of healthy
offspring.
Other problems in clinical applications of IVD gametes
are the immunologic and genetic challenges of stem cell
therapy. Application of IVD gametes derived from autologous stem cells provides a solution to overcome this problem. Mesenchymal stem cells and induced pluripotent stem
(iPS) cells are promising sources for the derivation of
gametes. Previously, direct differentiation of skin cells to
germ cells was shown.32–34 We demonstrated clearly that
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iPS cells derived from skin fibroblasts are able to differentiate to early stages of germ cells and express specific markers
for primordial germ cells (►Fig. 2). Derivation of germ cells
from iPS cells of infertile patients and comparing them with
those from normal individuals also offers an excellent
system to study pathologic germ cell specification and
differentiation. This would be a major advance in developing a model to elucidate the causes of infertility and other
reproductive disorders that are initiated during embryonic
development.
IVD Sperm as a Model for Nanorobots
Another important aspect of IVD sperm is its application as a
mechanical model in developing medical nanorobots. The
nanomechanical systems for medical applications (e.g., drug
delivery system for the treatment of cancer patients) will
significantly expand the effectiveness, precision, and speed of
future medical treatments while at the same time significantly reducing their risk, cost, and invasiveness. Sperm-like
derived cells exhibit the formation of flagella. Flagella comprise the biological apparatus necessary for movement and
penetration of the egg and therefore can be used as means to
drive the nanorobots.
Many aspects of flagellar motion have been studied, but
none of these studies have determined the exact equation for
the flagella abnormalities. Most of them are restricted to
linear theory or weakly nonlinear theories35 and are based on
small deformation assumptions. Because the deformation of
flagellum in nano-scale is very large with respect to its own
size, the small deformation assumption does not hold. To
study the dynamics, stability, and deformation of flagella, a
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West et al.
Figure 2 Stem cells derived from skin differentiated to germ cells. Human-induced pluripotent stem cells (hiPSCs) were differentiated for 10 days
on mouse embryonic fibroblast feeder cells in 20% KSR media. This system relies on spontaneous germ cell signaling in culture as a result of not
passaging and every other day media changes as previously done in our laboratory. 22–24 (A) Flow cytometry revealed a significant ( statistically
significant relative to iPSCs; p < 0.05) increase in the number of DDX4/POU5F1þ (22.75%) oocyte-like cells (OLCs) postdifferentiation when
compared with undifferentiated counterparts (0.95%). Day 10 differentiation cultures also demonstrated significant ( statistically significant
relative to iPSCs; p < 0.05) upregulation of the germ cell premigratory genes IFITM3 and KIT, migratory genes DAZL and DDX4, the postmigratory
genes PIWIL2 and NANOS1, and the meiotic gene SYCP1.
geometrically exact nonlinear model with initial twist and
curvature must be considered. Sperm whose locomotion is
empowered by propelling are tiny examples analogous to
large-scale vehicles that are used and studied in aerospace.
Rotorcrafts such as helicopters develop the required force for
locomotion in a way analogous to the way a sperm does—but
in a different direction. Multidisciplinary studies combining
biology, mechanics, and electronics may help us to design an
appropriate model for developing biological apparatuses for
developing nanorobots.
6 Toyooka Y, Tsunekawa N, Akasu R, Noce T. Embryonic stem cells
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8
9
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